PAX5 is one of the key transcription factors mediating differentiation of B-lymphocytes. It transcriptionally activates and represses a very large number of genes that permits the development of B-lymphocytes and prevents differentiation to T-lymphocytes or myeloid cells. We examined by SNP chip 633 acute lymphocytic leukemia (ALL) samples for PAX5 alterations (469 pediatric cases, 70 pediatric relapse cases, 74 adult cases and 50 ALL samples growing as xenografts). PAX5 genomic abnormalities occurred in H 27% of the samples including 26 PAX5 fusions to one of 5 other genes. Overall goal of the grant is to understand the clinical and pathologic significance of PAX5 alterations in ALL. Specific Aim 1 will determine frequency of genomic abnormalities of PAX5 in ALL and determine their clinical impact. Specific Aim 2 will define and understand the aberrant functions of PAX5 fusion and mutant proteins in ALL (Ex Vivo Studies). Studies will include gel retardation and reporter gene analysis as well as testing the ability of these proteins transcriptionally to activate selected target genes. Detailed studies will be done using two of the PAX5 fusions [PAX5-ETV6; PAX5- C20orf112 (C20)] including genome-wide identification of target genes of PAX5 fusion proteins in ALL using cDNA microarray analysis and high through-put ChIP sequencing studies. Comprehensive validation of the results will use a variety of techniques. Also, effect of PAX5 fusion proteins on hematopoietic cell differentiation will be determined. Specific Aim 3 will use in vivo models to examine the aberrant function of PAX5 fusions and deletions. First, we will determine if expression of PAX5 fusion proteins disrupts normal steady-state lymphopoiesis or hematopoiesis by impairing differentiation, promoting survival and/or proliferation of specific compartments? Second, we will identify secondary events that synergize with PAX5 fusion proteins to induce ALL. Third, we will determine if PAX5 deletions affect the course of human Ph1+ ALL xenografts. In summary, we will for the first time, correlate PAX5 alterations with clinical and pathological characteristics of the patients and define fully the functional significance of these alterations. These studies will have importance for classification of the disease, offer new therapeutic targets and foster our understanding of the pathogenesis of ALL.